To understand how genes and the products they encode are differentially regulated in response to environmental and physiological changes, one must elucidate the molecular mechanisms that govern the regulatory networks and signal transduction pathways of the cell. The nitrate assimilation pathway in plants provides a versatile system for studying such mechanisms. Nitrate assimilation involves the uptake and then reduction of nitrate to ammonia by the enzymes nitrate reductase (NR) and nitrite reductase (NiR). The transport systems and reductases in this pathway and the genes that encode them are tightly controlled so that they respond to several key environmental, metabolic and hormonal signals. The mechanisms that control nitrate metabolism include transcription regulation and a phosphorylation-dependent inhibition of NR by 14-3-3 proteins. Our long range goal is to elucidate further the regulatory mechanisms and to identify and characterize the genes required for nitrate uptake and reduction. To pursue this objective, we propose to continue our studies of nitrate metabolism in the plant Arabidopsis thaliana. A nitrate transporter gene CHLI has been cloned, characterized and shown to be one of at least two components of the low affinity nitrate uptake system. Subsequently, chlorate resistant mutants in two distinct complementation groups (CHL8 and CHL9) that are defective in high affinity nitrate uptake were isolated and studied. Our work on nitrate uptake will be continued by isolating and characterizing the CHL8 and CHL9 genes and determining their expression patterns and role/function in nitrate uptake, determining biochemical properties of the CHL8 and CHL9 gene products, determining the role CHL1 plays in high affinity uptake, and continuing our search for nitrate transport mutants. This work will further elucidate the mechanisms and regulation of nitrate transport. Our work on the regulation of nitrate reductase has advanced by the adaptation of two yeast (Pichia and two- hybrid) expression systems to study the phosphorylation-dependent regulation of NR by 14-3-3 proteins. The elucidation of the regulatory mechanisms and genes required for nitrate reduction will be continued by delineating the motifs and residues in both NR 14-3-3 proteins that are required for phosphorylation-dependent regulation of NR, determining the importance of these motifs on NR activity and regulation in vitro and in vivo, and identifying nitrate regulatory mutants using transposon mutagenesis. These studies have and will aid in the detection and reduction of nitrate, which is a prevalent health hazard in food and drinking water supplies.